Hepatitis C virus (HCV) is a leading cause of chronic hepatitis and hepatocellular carcinoma worldwide and infects more than 1% of the world?s population. Successful vaccine development is pivotal in controlling this global health problem. A system for efficient assembly of HCV structural proteins into HCV-like particles (VLPs) in insect cells has been developed in our laboratory. These noninfectious HCV-like particles had similar morphologic, serologic and biophysical properties as the putative virions isolated from HCV infected humans. In contrast to recombinant subunit vaccines, the viral proteins of HCV-like particles may be presented in a native, virion-like conformation and may therefore be superior in eliciting a protective humoral and cellular immune response. Using HCV-LP as a capture antigen in an enzyme-linked immunosorbant assay (ELISA), anti-HCV antibodies were specifically detected in serum from individuals with acute and chronic HCV infection. The humoral and cellular immunogenicity of the viruslike particles with or without viral p7 protein was studied. The HCV-LPs were inoculated intraperitoneally without any adjuvant into either BALB/c or AAD (C57BL/6 transgenic for HLA-A2.1) mice. Immunized mice developed high titers of anti-E2 antibodies and virus-specific cellular immune responses including cytotoxic T lymphocytes and T helper responses with gamma interferon production. The viruslike particles without p7 generated a higher cellular immune response with a more TH1 profile than the particles with p7. Immunization of heat denatured particles resulted in substantially lower humoral and cellular responses, suggesting that the immunogenicity is strongly dependent on particle formation. To evaluate the potential of HCV-LPs as a protective immunogen in a surrogate model, we challenged HCV-LP-immunized mice one month after the last boost with a recombinant vaccinia virus expressing core, E1 and E2 (vvHCV-ST). Mice immunized with viruslike particles were protected from challenge with the recombinant vvHCV.S (> 5 log decrease in titer) but not the control virus (vvlacZ). In comparison, DNA immunization with a construct expressing the HCV structural genes resulted in much less protection from HCV-vaccinia challenge (1-2 log decrease in titer). Adoptive transfer of splenocytes from immunized to nonimmunized mice conferred protection and selective depletion of CD4 or CD8 population abrogated the protective immunity. Our results indicate that hepatitis C viruslike particles can induce humoral and cellular immune responses that are protective in a surrogate animal model and offers a promising approach to vaccine development. We plan to conduct experiments in chimpanzees to test the effectiveness of the HCV-LP as a vaccine candidate.